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Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
The majority of mammalian cells that are cultured in vitro in petri dishes, flasks, and bioreactors adhere to the substrate surfaces by a sequence of steps that includes an initial attachment, flattening and spreading (Sagvolden et al., 1999; Khalili and Ahmad, 2015). Furthermore, cell to cell adhesion also occurs as proliferation proceeds. Integrin receptors and heterodimeric transmembrane proteins play a central role in this process of cell-substrate and cell-cell adhesion (Sagvolden et al., 1999). Following proliferation and once growth is halted, for example at a certain degree of confluence, cells must be dissociated from each other and from the surfaces of the containers or microcarriers used as supports, for further processing or passaging to a second stage of proliferation. The use of enzymes in these harvesting and sub-culturing operations is widespread and well known to researchers working with mammalian cells. These operations rely essentially on trypsin (EC 3.4.21.4), a serine endopeptidase found in the digestive system of many vertebrates, which preferentially hydrolyzes proteins by cleaving bonds next to arginine and lysine residues (Stroud et al., 1974). Trypsin is used specifically to hydrolyze the proteins involved in cell adherence, and thus promote disaggregation and detachment from the substratum (Gori, 1964; Codinach et al., 2016). The procedure is called trypsinization and can be found in processes used to manufacture recombinant proteins (Spearman et al., 2005), viral vectors (Fernandes et al., 2013), advanced therapy medicinal products (Koller et al., 1993; Stute et al., 2005; Kami et al., 2013; Codinach et al., 2016), vaccines (Wu et al., 2005), or other cell culture–derived medicinal products (Sun and Zhang, 2007; Sinacore et al., 2000).
Development of clone with novel TrpE fusion tag in E. coli for overexpression of trypsin in a bench-scale bioreactor
Published in Preparative Biochemistry & Biotechnology, 2021
Santhosh Nagaraj Nanjundaiah, Jayasri MA, Sunilkumar Sukumaran, Ganesh Sambasivam
Trypsin (EC 3.4.21.4) is a highly valuable serine protease of molecular weight 23.3 kDa, which targets basic amino acids such as lysine and arginine at the C-terminus. The zymogen form of the enzyme called trypsinogen gets converted to trypsin by the addition of either trypsin or enterokinase. Trypsin plays a major role in metabolism, digestion and coagulation in mammals.[1] Besides, the enzyme is useful in leather bating, food processing, pharmaceuticals and clinical diagnosis.[2] The application of trypsin in cell culture mainly lies in the removal of adherent cells from the culture surface and in the resuspension of cells.[3] The optimal pH for trypsin activity is 7–9, hence the formulation buffer should be of acidic pH to prevent self-activation.[4] To date, trypsin used in the laboratory as well as on the commercial scale is obtained from bovine and swine pancreas. Nonetheless, if extracted from these sources, the risk of microbial load being carried over even after the purification step is high.[5] The use of recombinant enzymes can help to overcome this complication. The bovine trypsin gene has been widely expressed in both prokaryotes and eukaryotes. Even though proteins are expressed without any fusion tag, the use of such tags are desirable as the enzymes are highly prone to degradation even when expressed in prokaryotic hosts. Fusion tags increase enzyme stability. However, the size of the tag is an important factor as it determines the final yield. The tag-protein ratio when using glutathione S transferase, maltose binding protein and thioredoxin fusion tag with trypsin is about 1:1–1:3. Although the expression levels are high, the total product percentage is only around 33–50% of the inclusion bodies produced. Besides, some amount of the protein is present in the soluble fraction, resulting in the reduction of yield after the purification and refolding steps. In order to convert the soluble fraction of protein into insoluble fraction, the use of an insoluble fusion partner with a hydrophobic core is desirable. In this context, the use of fusion tags that are less than 2 kDa in size and a ratio of up to 1:8–1:10 (tag: protein) might reduce the cost of production by increasing the protein yield. This approach could result in 80–90% of the protein being pushed into the inclusion bodies fraction.